The present invention relates to a method and apparatus for the sequential degradation of protein or amino acid chains, specifically by the Edman technique.
At the present time, the sequencing of peptides and proteins is most commonly performed by a series of chemical reactions which, in one cycle of the sequencing process, is referred to as the Edman degradation. Briefly summarized, a typical Edman degradation includes the steps of coupling, cleavage and conversion. The peptide of N amino acids in length is coupled to coupling reagent, phenylisothiocyanate (PITC), in a basic environment to form a phenylthiocarbamyl (PTC) derivative of the peptide. During cleavage, the PTC-peptide derivative is treated with a cleavage reagent, strong anhydrous acid, to form an unstable cyclic derivative of the peptide which rapidly cleaves into the more stable anilinothiazolinone (ATZ) derivative of the amino-terminal amino acid and a free peptide or amino acid of N-1 length which is the original peptide with the terminal amino acid removed. The final stage of the process, conversion, is performed manually away from conventional automated sequencing apparatus. The ATZ-amino acids produced by the Edman degradation, which are highly unstable in the presence of oxygen, are treated with aqueous acid to convert them into the more stable phenylthiohydantoin amino acids (PTH-amino acids) for chromatographic identification. The shortened peptide or protein is employed as a starting material for another cycle of the Edman degradation.